Biochemistry, Biophysics, and Structural Biology Commons^™

Kinetic, Mechanistic, And Structural Investigation Of Features Controlling Stereoselectivity Of (R)- And (S)-Hydroxypropyl Com Dehydrogenases From Xanthobacter Autrophicus Strain Py2, Dariusz Adam Sliwa

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Enantiopure alcohols are valuable intermediates in fine organic synthesis, in particular for preparation of biologically active compounds. The necessity of preparing single enantiomer drugs in an optically pure form has triggered much research, especially in the pharmaceutical industry. The biocatalytical production of chiral alcohols by alcohol dehydrogenase enzymes is characterized by the asymmetric reduction of the corresponding ketones, usually with high degree of stereoselectivity. The commercial value of the enzymes as stereoselective biocatalysts has been a significant driving force in understanding features that control their mechanism of catalysis and stereoselectivity. This work focuses on two enantiocomplementary dehydrogenase enzymes ((R …

Understanding Gafp, A Plant Lectin With Broad Spectrum Inhibitory Activity, Alexis Nagel

All Dissertations

South Carolina and Georgia are the largest peach producing regions in the Southeastern United States, generating about $60 million worth (~90,000 tons) of fruit per year on average. Peaches and other stone-fruits (Prunus sp.) can be afflicted by a variety of root-associated diseases which negatively impact annual yield and long-term tree mortality. An engineered Prunus rootstock with enhanced resistance to soil-borne pathogens would therefore be of great benefit to the Southeastern peach industry. The Gastrodia anti-fungal protein (GAFP) is a monocot mannose-binding lectin which is able to inhibit the growth of multiple species of plant pathogenic fungi. Previous findings from …

Spidroin N-Terminal Domain: A Ph Sensor In The Spider Silk Assembly Process, William Gaines

All Dissertations

Spider silks are protein-based fibers with remarkable mechanical qualities. Perhaps even more impressive is the spinning process in which the spider silk proteins (spidroins) are assembled from a highly soluble storage state into a well-ordered and insoluble fiber. Indeed, the ordered arrangement of spidroins, which is endowed by the spinning process, is the basis of fiber strength. However, the forces driving fiber assembly and the mechanisms by which spidroins respond those forces are only poorly understood. Spidroins have a tripartite domain architecture consisting of a large and repetitive central domain flanked by small, non-repetitive N- and C-terminal domains. Both terminal …

Nitrosative Stress And Dna Repair In Microorganisms, Sung-Hyun Park

All Dissertations

DNA can be damaged by reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) from both endogenous and exogenous sources. Under nitrosative stress conditions, DNA may be modified through base deamination or crosslinking. Uracil (U), xanthine (X) and oxanine (O), hypoxanthine (I), and thymine (T) are the corresponding deamination products derived from cytosine (C), guanine (G), adenine (A), and 5-methylcytosine, respectively. To remove DNA damage, cells are equipped with a variety of DNA repair enzymes which participate in different repair pathways.
My dissertation work involves studies of genes and enzymes in nucleotide excision repair (NER) and base excision repair (BER) …

Excision Dynamics Of Vibrio Pathogenicity Island-2 From Vibrio Cholerae: Role Of A Recombination Directionality Factor Vefa, Salvador Almagro-Moreno, Michael G. Napolitano, E. Fidelma Boyd

Dartmouth Scholarship

Vibrio Pathogenicity Island-2 (VPI-2) is a 57 kb region present in choleragenic V. cholerae isolates that is required for growth on sialic acid as a sole carbon source. V. cholerae non-O1/O139 pathogenic strains also contain VPI-2, which in addition to sialic acid catabolism genes also encodes a type 3 secretion system in these strains. VPI-2 integrates into chromosome 1 at a tRNA-serine site and encodes an integrase intV2 (VC1758) that belongs to the tyrosine recombinase family. ntV2 is required for VPI-2 excision from chromosome 1, which occurs at very low levels, and formation of a non-replicative circular intermediate.

Requirement For Golgi-Localized Pi(4)P In Fusion Of Copii Vesicles With Golgi Compartments, Andres Lorente-Rodriguez, Charles Barlowe

Dartmouth Scholarship

The role of specific membrane lipids in transport between endoplasmic reticulum (ER) and Golgi compartments is poorly understood. Using cell-free assays that measure stages in ER-to-Golgi transport, we screened a variety of enzyme inhibitors, lipid-modifying enzymes, and lipid ligands to investigate requirements in yeast. The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport. Analysis of wild type and mutant PH domain proteins in addition to recombinant versions of the Sac1p phosphoinositide-phosphatase indicated that PI(4)P was required on Golgi membranes for fusion with coat protein complex II (COPII) …

An Archaeal Trna-Synthetase Complex That Enhances Aminoacylation Under Extreme Conditions, Vlatka Godinic-Mikulcic, Jelena Jaric, Corinne D. Hausmann, Michael Ibba, Ivana Weygand-Durasevic

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translation, although the precise biological role remains largely unknown. To gain further insights into archaeal MSCs, possible protein-protein interactions with the atypical Methanothermobacter thermautotrophicus seryl-tRNA synthetase (MtSerRS) were investigated. Yeast two-hybrid analysis revealed arginyl-tRNA synthetase (MtArgRS) as an interacting partner of MtSerRS. Surface plasmon resonance confirmed stable complex formation, with a dissociation constant (KD) of 250 nm. Formation of the MtSerRS·MtArgRS complex …

Systematic Approach To Electrostatically Induced 2d Crystallization Of Nanoparticles At Liquid Interfaces, Sumit Kewalramani, Suntao Wang, Yuan Lin, Huong Giang Nguyen, Qian Wang, Masafumi Fukuto, Lin Yang

Faculty Publications

We report an experimental demonstration of a strategy for inducing two-dimensional (2D)crystallization of charged nanoparticles on oppositely charged fluid interfaces. This strategy aims to maximize the interfacial adsorption of nanoparticles, and hence their lateral packing density, by utilizing a combination of weakly charged particles and a high surface charge density on the planar interface. In order to test this approach, we investigated the assembly of cowpea mosaic virus (CPMV) on positively charged lipid monolayers at the aqueous solution surface, by means of in situX-ray scattering measurements at the liquid–vapor interface. Theassembly was studied as a function of the solution …

Work Towards The Isolation And Characterization Of The Muscle Isoform Of Glucose 1,6-Bisphosphatase, Caleb J. Hiller

Theses and Dissertations

Glucose 1,6-bisphosphate is an important small molecule involved in the regulation of glycolysis. Four enzymes synthesize this compound. One enzyme is known to degrade it, glucose 1,6-bisphosphatase. Other groups have produced work that indicates that there are two isoforms of this enzyme, one predominant in the brain and one in the muscle. This thesis contains the work performed in attempts to isolate and characterize the muscle isoform of glucose 1,6-bisphosphatase. While this enzyme was not isolated, much was learned about it and the results from this work may help in the future identification of this enzyme.

Leishmania Major Survival In Selective Phlebotomus Papatasi Sand Fly Vector Requires A Specific Scg-Encoded Lipophosphoglycan Galactosylation Pattern, Deborah E. Dobson, Shaden Kamhawi, Phillip Lawyer, Salvatore J. Turco, Stephen M. Beverley, David L. Sacks

Molecular and Cellular Biochemistry Faculty Publications

Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In "selective" sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the "selective" fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated …

Resonance Assignments And Secondary Structure Predictions Of The As(Iii) Metallochaperone Arsd In Solution, Jun Ye, Yanan He, Jack Skalicky, Barry P. Rosen, Timothy L. Stemmler

Biochemistry and Molecular Biology Faculty Publications

ArsD is a metallochaperone that delivers As(III) to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Conserved ArsD cysteine residues (Cys12, Cys13 and Cys18) construct the As(III) binding site of the protein, however a global structural understanding of this arsenic binding remains unclear. We have obtained NMR assignments for ArsD as a starting point for probing structural changes on the protein that occur in response to metalloid binding and upon formation of a complex with ArsA. The predicted solution structure of ArsD is in agreement with recently published …

Aminopeptidases Do Not Directly Degrade Tau Protein, K. Martin Chow, Hanjun Guan, Louis B. Hersh

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: Tau hyperphosphorylation and aggregation to form intracellular neurofibrillar tangles is prevalent in a number of tauopathies. Thus there is current interest in the mechanisms involved in Tau clearance. It was recently reported that Tau can be degraded by an aminopeptidase known as the puromycin sensitive aminopeptidase (PSA). Until now PSA has been reported to only cleave peptides, with the largest reported substrates having 30-50 amino acids. We have studied this unique PSA cleavage reaction using a number of different PSA preparations.

RESULTS: An N-terminally His tagged-PSA was expressed and purified from Sf9 insect cells. Although this PSA preparation cleaved …

The Mycobacterium Tuberculosis Drugome And Its Polypharmacological Implications, Sarah L. Kinnings, Li Xie, Kingston H. Fung, Richard M. Jackson, Lei Xie, Phillip E. Bourne

Publications and Research

We report a computational approach that integrates structural bioinformatics, molecular modelling and systems biology to construct a drug-target network on a structural proteome-wide scale. The approach has been applied to the genome of Mycobacterium tuberculosis (M.tb), the causative agent of one of today’s most widely spread infectious diseases. The resulting drug-target interaction network for all structurally characterized approved drugs bound to putative M.tb receptors, we refer to as the ‘TB-drugome’. The TB-drugome reveals that approximately one-third of the drugs examined have the potential to be repositioned to treat tuberculosis and that many currently unexploited M.tb receptors may be chemically druggable …

Dnmt1 Stability Is Regulated By Proteins Coordinating Deubiquitination And Acetylation-Driven Ubiquitination, Zhanwen Du, Jing Song, Yong Wang, Yiqing Zhao, Kishore Guda, Shuming Yang, Hung Ying Kao, Yan Xu, Joseph Willis, Sanford D. Markowitz, David Sedwick, Robert M. Ewing, Zhenghe Wang

Chemistry Faculty Publications

DNA methyltransferase 1 (DNMT1) is the primary enzyme that maintains DNA methylation. We describe a previously unknown mode of regulation of DNMT1 protein stability through the coordinated action of an array of DNMT1-associated proteins. DNMT1 was destabilized by acetylation by the acetyltransferase Tip60, which triggered ubiquitination by the E3 ligase UHRF1, thereby targeting DNMT1 for proteasomal degradation. In contrast, DNMT1 was stabilized by histone deacetylase 1 (HDAC1) and the deubiquitinase HAUSP (herpes virus–associated ubiquitin-specific protease). Analysis of the abundance of DNMT1 and Tip60, as well as the association between HAUSP and DNMT1, suggested that during the cell cycle the initiation …

Halo Ion Trap Mass Spectrometry: Design, Instrumentation, And Performance, Miao Wang

Theses and Dissertations

New ion trap mass spectrometry (ITMS) instrumentation, the toroidal IT and halo IT, were developed to meet the significant growth in on-site analysis applications. The miniature toroidal IT mass analyzer was operated with radio frequency (RF) trapping voltages of 3 kVp-p or less. Despite its reduced dimensions, it has roughly the same ion trapping capacity as conventional 3D quadrupole ITs. Unit-mass resolved spectra for n-butylbenzene, xenon, and naphthalene were obtained. The desired linear mass scale was obtained using conventional mass-selective instability scan combined with resonance ejection. The halo IT was also based on toroidal trapping geometry and microfabrication technology, consisting …

Evaluation Of Immunogene Therapy Using A Plasmid Encoding Il-15 Delivered By Electroporation In A 3d Tumor Model And A Mouse Melanoma Model, Bernadette Marrero

USF Tampa Graduate Theses and Dissertations

Melanoma is an aggressive disease with few effective treatment options. Non-toxic, anti-tumor therapies and prophylactic approaches are currently being investigated to identify treatment options that will control and remove late-stage melanoma.

The overall goal of this project was to establish an effective delivery method for a plasmid encoding human interleukin (phIL-15) into mouse melanoma cells (B16.F10) using the gene transfer technique electroporation (EP)¹. The EP delivery phIL-15 was optimized using an in vitro 3D tumor model. The purpose was to translate these IL-15 delivery conditions into an in vivo mouse melanoma model to study IL-15 signal transduction and …

3,4-Methylenedioxymethamphetamine Alters Left Ventricular Function And Activates Nuclear Factor-Kappa B (Nf-Kappab) In A Time And Dose Dependent Manner, David A. Tiangco, Sapna Halcomb, Frank A. Lattanzio Jr, Barbara Y. Hargrave

Bioelectrics Publications

3,4-Methylenedioxymethamphetamine (MDMA) is an illicit psychoactive drug with cardiovascular effects that have not been fully described. In the current study, we observed the effects of acute MDMA on rabbit left ventricular function. We also observed the effects of MDMA on nuclear factor-kappa B (NF-kappa B) activity in cultured rat ventricular myocytes (H9c2). In the rabbit, MDMA (2 mg/kg) alone caused a significant increase in heart rate and a significant decrease in the duration of the cardiac cycle. Inhibition of nitric oxide synthase (NOS) by pretreatment with L-NAME (10 mg/kg) alone caused significant dysfunction in heart rate, systolic pressure, diastolic pressure, …

Andrews Student Named 2010 American Chemical Society Scholar, Dave Randall

Lake Union Herald

No abstract provided.

Virulence Of An Emerging Respiratory Pathogen, Genus Pandoraea, In Vivo And Its Interactions With Lung Epithelial Cells, Gillian Herbert, Anne Costello, Lydia Fabunmi, Kirsten Schaffer, Kevin Kavanagh, Emma M. Caraher, Máire Callaghan, Siobhan Mcclean

Articles

Pandoraea species have emerged as opportunistic pathogens among cystic fibrosis (CF) and non-CF patients. Pandoraea pulmonicola is the predominant Pandoraea species among Irish CF patients. The objective of this study was to investigate the pathogenicity and potential mechanisms of virulence of Irish P. pulmonicola isolates and strains from other Pandoraea species. Three patients from whom the P. pulmonicola isolates were isolated have since died. The in vivo virulence of these and other Pandoraea strains was examined by determining the ability to kill Galleria mellonella larvae. The P. pulmonicola strains generally were the most virulent of the species tested, with three …

Retention And Loss Of Rna Interference Pathways In Trypanosomatid Protozoans, Lon-Fye Lye, Katherine Owens, Huafang Shi, Silvane M. F. Murta, Ana Carolina Vieira, Salvatore J. Turco, Christian Tschudi, Elisabetta Ullu, Stephen M. Beverley

Molecular and Cellular Biochemistry Faculty Publications

RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the …